Journal: bioRxiv

Article Title: Suppression of non-canonical autophagy induces endothelial and cardiac dysfunction

doi: 10.64898/2025.12.17.695030

Figure Lengend Snippet: A . Recycled VEGFR2 stained with Alexa674 (Cyan) and nuclei stained with Hoechst (Red) in human aortic endothelial cells (HAEC) RUBCN KO and RUBCN WT exposed to static or 5 dynes of laminar flow during 24h. Scale bars, 1mm. Images are representative of three independently performed experiments. B. Volume of recycled VEGFR2 per cell with each point representing the average volume for at least 3 cells per field view from HAEC RUBCN KO and RUBCN WT (n=3 independent replicates per group) exposed to static or 5 dynes of laminar flow during 24h. IgG controls as a control for VEGFR2 antibody. Values are expressed as mean±SEM. Significance was calculated for the Volume of recycled VEGFR2 with a 2-way ANOVA followed by tukey’s multiple comparison test (Flow exposure effect: *P<0.05 ; Genotype effect: ****P<0.0001; Flow exposure Genotype effect: *P<0.05 ). C. Volume of recycled VEGFR2 per cell with each point representing the average volume for at least 3 cells per field view from murine PMVEC isolated from 2-month-old Atg16l1 wt mice n=3 and Atg16l1 DWD ki mice n=3 then exposed to static or 5 dynes of laminar flow during 24h. Values are expressed as mean±SEM. Significance was calculated for the volume of VEGFR2 recycled per cell with an unpaired Student t test . *P<0.05 D. Representative immunoblots of VEGFR2 and actin in isolated surface, whole-cell lysate (WCL) and biotin unbound fractions across murine PMVEC isolated from 2-month-old Atg16l1 wt and Atg16l1 DWD ki mice then exposed to static or 5 dynes of laminar flow during 24h. E. Immunoblots of total protein S-nitrosylation in whole cell lysate from PMVEC isolated from 2-month-old Atg16l1 wt mice n=3 and Atg16l1 DWD ki mice n=3, then exposed to static or 5 dynes of laminar flow during 24h. F. Total protein S-nitrosylation densitometry measured by western blot in PMVEC isolated from 2-month-old Atg16l1 wt mice n=3 and Atg16l1 DWD ki mice n=3 then exposed to static or 5 dynes of laminar flow during 24h. Values are expressed as mean±SEM. Significance was calculated for the densitometry with a 2-way ANOVA followed by tukey’s multiple comparison test. G. Nitrites measured in cell culture medium in HAEC RUBCN KO and RUBCN WT (n=3 independent replicates per group) exposed to static or 5 dynes of laminar flow during 24h.Values are expressed as mean±SEM. Significance was calculated for the nitrites with a 2-way ANOVA followed by tukey’s multiple comparison test (Flow exposure effect: **P<0.01 ; Genotype effect: *P<0.05 ). H. Immunoblots of total VEGFR2, Y1175-phosporylated-VEGFR2, Ser1177-phosphorylated-eNOS, total eNOS and b-actin proteins in whole cells lysates of HAEC RUBCN KO and RUBCN WT (n=3 independent replicates per group) exposed to static or 5 dynes of laminar flow during 24h.

Article Snippet: Human aortic endothelial cells (HAEC) were purchased at ATCC (Cat#PCS-100-011), plated on fibronectin coated plates (1μg/cm , Corning; Cat# 354008) and maintained in EBM-2 medium (Lonza, Cat# CC-3156) completed with Endothelial Cell growth medium-2 bullet kit (Lonza, Cat# CC-3162) in a humidified 5% CO chamber at 37°C.

Techniques: Staining, Control, Comparison, Isolation, Western Blot, Cell Culture

Journal: American journal of physiology. Heart and circulatory physiology

Article Title: COL8A1 Regulates Endothelial Phenotype in Inflammatory Endothelial-to-Mesenchymal Transition

doi: 10.1152/ajpheart.00339.2025

Figure Lengend Snippet: (A) Brightfield images of HAECs after TNF-α treatment (50 ng/mL), showing morphological changes. (Scale bar: 50 μm). (B) Immunofluorescence staining for PECAM1 (green) and N-cadherin (red) in control or TNF-α-treated HAECs for 96 hours. Nuclei were counterstained with DAPI (blue). (Scale bar: 50 μm). (C) RT-qPCR of endothelial markers ( PECAM1 and ENOS3 ) and mesenchymal markers ( CDH2, COL1A1, COL3A1, FN1 , and CNN1 ) in HAECs at various timepoints (24, 48, and 96 hours). Analysis by two-tailed Student’s t -test. (D) In vitro Matrigel-based tube formation assays of control or TNF-α-treated (50 ng/mL) HAECs for 96 hours. Cells were labeled with CellTracker Green CMFDA. (Scale bar: 200 μm) (E) Total tube length and Number of nodes in each group for the tube formation assay (n = 4). The values are expressed as mean±SEM, analysis by two-tailed Student’s t -test.

Article Snippet: Primary human aortic endothelial cells were purchased from (HAECs; Cell Applications, Cat# 211-500, Lot #2986), all experiments in this study were performed using cells from the same lot to avoid batch-to-batch variation.

Techniques: Immunofluorescence, Staining, Control, Quantitative RT-PCR, Two Tailed Test, In Vitro, Labeling, Tube Formation Assay

Journal: American journal of physiology. Heart and circulatory physiology

Article Title: COL8A1 Regulates Endothelial Phenotype in Inflammatory Endothelial-to-Mesenchymal Transition

doi: 10.1152/ajpheart.00339.2025

Figure Lengend Snippet: (A) Matrigel-based tube formation assays of HAECs transfected with control (siCtrl) or COL8A1 siRNA (siCOL8A1), with or without TNF-α treatment for 4 days (scale bar, 200 μm). (B) Quantification of total tube length and number of nodes in each group (n = 5). Data are mean ± SEM, statistical analysis by one-way ANOVA. (C-E) Bulk RNA-seq datasets of control or COL8A1 downregulated HAECs, both of which treated with control siRNA or COL8A1 siRNA for 24 hours. (C) Gene Ontology (GO)-Molecular Function (MF) analysis showing the pathway enrichments. (D) Volcano plot, showing differentially expressed genes (defined as fold-change>2, p value<0.05) upon COL8A1 knockdown (siCOL8A1 vs. siCtrl). The annotated genes are COL8A1, and key genes related to endothelial function. (E) Gene Set Enrichment Analysis (GSEA), demonstrating Epithelial to Mesenchymal transition as positive enriched pathway related to COL8A1 knockdown and the differentially expressed genes contributing to the pathway. (F) Western blot analysis of COL8A1, PECAM1, eNOS3, TAGLN, Snail, and GAPDH (loading control) in HAECs transfected with Control siRNA, COL8A1 siRNA, or treated with TNF-α for 60 hours. (G) Quantification of normalized protein expression levels for COL8A1 (15 kDa and 85 kDa), PECAM1, TAGLN, and Snail, relative to GAPDH (n = 3). Data are mean ± SEM. (H) HAECs transfected with siCtrl or siCOL8A1 were cultured on BSA- or rcol8a1-coated plates. RT-qPCR analysis of PECAM1, ENOS3, and CDH2 expression (n = 6). Data are mean ± SEM; statistical analysis by two-tailed Student’s t-test.

Article Snippet: Primary human aortic endothelial cells were purchased from (HAECs; Cell Applications, Cat# 211-500, Lot #2986), all experiments in this study were performed using cells from the same lot to avoid batch-to-batch variation.

Techniques: Transfection, Control, RNA Sequencing, Knockdown, Western Blot, Expressing, Cell Culture, Quantitative RT-PCR, Two Tailed Test

Journal: American journal of physiology. Heart and circulatory physiology

Article Title: COL8A1 Regulates Endothelial Phenotype in Inflammatory Endothelial-to-Mesenchymal Transition

doi: 10.1152/ajpheart.00339.2025

Figure Lengend Snippet: (A) RT-qPCR analysis of endothelial markers, including KDR (VEGFR2), ENOS3, PECAM1 , and mesenchymal ( ACTA2) markers in HAECs transduced with control or COL8A1 -overexpressing ( COL8A1 oe ) lentivirus and treated with TNF-α for 24 hours (n=3–4). Statistical analysis by one-way ANOVA, and the values are expressed as mean±SEM. (B) Tube formation assay of control or COL8A1 oe HAECs with or without TNF-α. The control lentivirus expressed mCherry protein (red) and the COL8A1 oe lentivirus expressed green fluorescent protein (green) (Scale bar: 200 μm). (C) Quantification of the number of junctions present in each group in the tube formation assay (n = 5). Statistical analysis by one-way ANOVA, and the values are expressed as mean±SEM.

Article Snippet: Primary human aortic endothelial cells were purchased from (HAECs; Cell Applications, Cat# 211-500, Lot #2986), all experiments in this study were performed using cells from the same lot to avoid batch-to-batch variation.

Techniques: Over Expression, Quantitative RT-PCR, Transduction, Control, Tube Formation Assay